The present invention is directed to a nucleic acid element that enhances gene expression in yeast in response to hypoxic conditions. This element may be ligated to genes to enhance recombinant protein production both in yeast and in other eukaryotic cells.
The ability to induce the expression of specific genes in response to hypoxia is a characteristic common to both prokaryotic and eukaryotic cells (Bunn, et al. Physiol. Rev. 76:839-885 (1996); Ebert, et al. Blood 94:1864-1877 (1999); Levy, et al. J. Biol. Chem. 270:13333-13340 (1995); Semenza, et al. Mol. Cell. Biol. 12:5447-5454 (1992)). Certain factors appear to mimic hypoxia and induce the expression of the same genes induced in response to low oxygen levels. These inducers include transition metals (e.g., cobalt and nickel) and iron chelators (Goldberg, et al. Science 42:1412-1415 (1988); Horiguchi, et al. Biochim. Biophys. Acta 1495:231-236 (2000); and Huang, et al. J. Biol. Chem. 271:32253-32259 (1996)). In addition, at least one factor, carbon monoxide, has been found to inhibit the expression of hypoxia-induced genes (Huang, et al. J. Biol. Chem. 274:9038-9044 (1999)).
Yeast such as Saccharomyces cerevisiae, respire in the presence of oxygen but ferment under anaerobic conditions. Not surprisingly, these organisms have evolved sophisticated molecular mechanisms involving oxygen dependent gene regulation. Several yeast genes, exemplified by ANB1, have been shown to be upregulated by complete anaerobiosis (Zitomer, et al. Microbiol Rev. 5:1-11 (1992)). In addition, other genes exhibit increased expression at low oxygen tensions, before complete anaerobic conditions are reached (Kwast, et al. Proc. Nat.""l Acad. Sci. USA 96:5446-5451 (1999)). The identification of the mechanisms by which yeast and other organisms regulate gene expression in response to low levels of oxygen should provide new insights into biological adaptations used for survival and new opportunities for controlling the cellular production of recombinant genes.
The present invention is based upon the discovery of a distinct hypoxia responsive enhancer element (HREE, also referred to herein as a low oxygen response element, or xe2x80x9cLORExe2x80x9d) that helps regulate the rate at which mRNA is transcribed from the OLE1 gene in yeast in response to low levels of oxygen. This element may be combined with other genes to increase recombinant protein production both in yeast and in other eukaryotic cells. The element responds both to low levels of oxygen (hypoxic conditions) and to essentially a complete absence of oxygen (anaerobic conditions). For the purposes of the present invention, reference to xe2x80x9chypoxiaxe2x80x9d or xe2x80x9chypoxic conditionsxe2x80x9d will include xe2x80x9canaerobic conditionsxe2x80x9d unless the context of usage indicates otherwise.
In its first aspect, the invention is directed to a substantially pure HREE DNA molecule consisting essentially of either the sequence ACYCAACAA (SEQ ID NO: 1) or GAACACYCAACAAACCTTAT (SEQ ID NO: 2). The symbol xe2x80x9cYxe2x80x9d indicates that the designated nucleotide may be either T or C. As used herein, the term xe2x80x9cconsisting essentially ofxe2x80x9d means that, starting with either SEQ ID NO: 1 or SEQ ID NO: 2, the invention includes minor changes in the sequences provided that such changes do not alter the basic biological characteristics of the elements with respect to their ability to induce gene expression in response to hypoxia and provided that the mutated product maintains at least a 50% structural homology to either SEQ ID NO: 1 or SEQ ID NO: 2. For example, mutated elements that evidenced a substantial (e.g. 70%) loss in their ability to induce expression would not be part of the invention. In this regard, it should be noted that SEQ ID NO: 1 forms a 9 nucleotide core within SEQ ID NO: 2 being flanked at its 5xe2x80x2 end by the sequence GAAC, and its 3xe2x80x2 end by the sequence CCTTAT. Core sequences joined to either all or part of these flanking sequences are encompassed by the invention. For example, SEQ ID NO: 1 may be flanked at its 5xe2x80x2 end by C, AC, AAC, or GAAC. Similarly, it may be flanked at its 3xe2x80x2 end by C, CC, CCT, CCTT, CCTA, or CTTAT.
The DNA sequence elements described above may be included in a vector for recombinantly expressing a peptide or protein in eukaryotic cell. The vector includes a promoter active in the eukaryotic cell, a hypoxia responsive enhancer element as described above, and a DNA sequence encoding the peptide or protein. The latter should be operably linked to the promoter. In addition, the sequence encoding the peptide or protein should be non-homologous to HREE. The term xe2x80x9cnon-homologousxe2x80x9d indicates that the HREE element is joined to a gene other than one it would normally be operably linked to in nature, e.g. the element must be joined to something other than the yeast OLE1 gene. The vector may be used to transform an appropriate host cell for the purpose of producing recombinant protein. Preferably, the host cell is a yeast and the promoter used in the vector is active in yeast cells. The most preferred promoter is the CYC1 basal promoter.
In another aspect, the invention is directed to a method for recombinantly producing a peptide or protein in which host cells transformed with the vector discussed above are grown under anaerobic conditions. In certain instances, such cells may have direct utility. For example, yeast cells might be transformed with a vector for the expression of a gene that enhances alcohol production during fermentation. In other instances, the method may include the purification of recombinant protein or peptide either directly from host cells or, preferably, from the medium surrounding the host cells. These methods for producing peptides or proteins may include exposing the transformed host cells to an agent that helps to induce recombinant gene expression. In particular, cells may be exposed to either transition metals or to iron chelators. The most preferred transition metals are cobalt and nickel.
The present invention also encompasses certain variations on the method for recombinantly producing peptides or proteins discussed above. In one embodiment, host cells transformed with an HREE-containing vector are first grown under aerobic conditions and recombinant expression of peptide or protein is then induced by exposing the cells to anaerobic conditions. Again, the preferred host cells are yeast and these may either be used directly or the recombinant protein can be purified prior to use.
In another variation, host cells are first grown under aerobic conditions and gene expression is then induced by exposing them either to a transition metal such as cobalt or nickel or by exposing them to an iron chelator. It is also possible to combine these methods so that the expression of genes is controlled both by changing oxygen availability and by altering the concentration of inducing agents.